Trophoblast glycoprotein (5T4, TPBG) specific chimeric antigen receptors for cancer immunotherapy

a trophoblast glycoprotein and immunotherapy technology, applied in the field of chimeric antigen receptors, can solve the problems of inability to provide prolonged expansion and anti-tumor activity in vivo, and cannot be expanded to any antigen

Active Publication Date: 2020-09-01
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]In the framework of the present invention, they have designed and implemented at 5T4 specific CAR having one of the polypeptide structure selected from V1 to V6 as illustrated in FIG. 2, said structure comprising an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-5T4 antibody, a hinge, a transmembrane domain and a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB. Preferred CAR polypeptides of the invention comprise an amino acid sequence selected from SEQ ID NO.19 to 42. Following non-specific activation in vitro (e.g. with anti CD3 / CD28 coated beads and recombinant IL2), T-cells from donors have been transformed with polynucleotides expressing these CARs using viral transduction. More preferred CAR polypeptides having a polypeptide structure selected from V3, V5, V1 (i.e. having the CD8a transmembrane domain) have shown the best and unexpected results.
[0015]The resulting engineered T-cells displayed reactivity in-vitro against 5T4 positive cells to various extend, showing that the CARs of the present invention contribute to antigen dependent activation, and also proliferation, of the T-cells, making them useful for immunotherapy.

Problems solved by technology

However, they failed to provide prolonged expansion and anti-tumor activity in vivo.
However, the particular combination of signaling domains, transmembrane and co-stimulatory domains used with respect to CD19 ScFv, was rather antigen-specific and cannot be expanded to any antigen markers.

Method used

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  • Trophoblast glycoprotein (5T4, TPBG) specific chimeric antigen receptors for cancer immunotherapy
  • Trophoblast glycoprotein (5T4, TPBG) specific chimeric antigen receptors for cancer immunotherapy
  • Trophoblast glycoprotein (5T4, TPBG) specific chimeric antigen receptors for cancer immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

example 1

tion of TCRalpha Inactivated Cells Expressing a 5T4-CAR

[0161]Heterodimeric TALE-nuclease targeting two 17-bp long sequences (called half targets) separated by an 15-bp spacer within T-cell receptor alpha constant chain region (TRAC) gene were designed and produced. Each half target is recognized by repeats of the half TALE-nucleases listed in Table 10

[0162]

TABLE 10TAL-nucleases targeting TCRalpha genePolynucleotidTargetTarget sequenceencode TALENHalf TALE-nucleaseTRAC_T01TTGTCCCACAGATATCCT01-LTRAC_T01-L TALENAgaaccctgaccctg(SEQ ID NO: 44)(SEQ ID NO: 46)CCGTGTACCAGCTGAGAT01-RTRAC_T01-R TALEN(SEQ ID NO: 43)(SEQ ID NO: 45)(SEQ ID NO: 47)

[0163]Each TALE-nuclease construct was subcloned using restriction enzyme digestion in a mammalian expression vector under the control of the T7 promoter. mRNA encoding TALE-nuclease cleaving TRAC genomic sequence were synthesized from plasmid carrying the coding sequence downstream from the T7 promoter.

[0164]Purified T cells preactivated during 72 hour...

example 2

of 5T4-Positive and -Negative Cell Line

[0167]Eight human cell lines were screened for 5T4 expression by western blot and flow cytometry (see Table 11 below).

[0168]

TABLE 11Expression of 5T4 antigen in 8 human cell linesCell lineDescriptionCell typeMCF7adherentadenocarcinomaHCT116adherentcolorectal carcinomaMKN45adherentgastric carcinomaLS174Tadherentcolorectal adecarcinomaSK-MEL-28adherentmalignant melanomaSupT1suspensionT-cell lymphoblastic lymphomaDaudisuspensionBurkitt's lymphoma

[0169]5T4 was not detected in extracts from Daudi (ATCC CCL-213), SupT1 (ATT CRL-1942) and SK-MEL-28 (ATCC HTB-72) cells but was detected in extracts from MCF7 (ATCC HTB-22), HCT116 (ATCC CCL-247), MKN45 (JCRB0254) and LS174T (ATCC CL-188) cells. Among the cells that were positive for 5T4 antigen following western blot analysis, only two were found to express 5T4 at the cell surface: MCF7 and HCT116 cells, MCF7 expressing highest levels of 5T4 antigen than HCT116 cells.

example 3

n of Anti-5T4 scCARs

[0170]Second generation singlechain CARs specific for 5T4 (shown schematically in FIGS. 3 to 6 and and in Table 3 to Table 6) were created by combining the sequences of 4 different scFv with the sequences of 3 different spacers, 2 different transmembrane domains, 1 costimulatory domain and 1 stimulatory domain as represented in FIG. 2.

[0171]The sequences used in the CARs (presented in Table 1 and Table 2) derive from:[0172]the H8, A1, A2 or A3 antibodies for the scFv;[0173]the IgG1, FcεRIIIγ or CD8α molecules for the spacer domain;[0174]the CD8α or 4-1BB molecules for the transmembrane domain;[0175]the 4-1BB molecule for the costimulatory domain;[0176]the CD3ζ molecule for the stimulatory domain.

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Abstract

The present invention relates to Chimeric Antigen Receptors (CAR) that are recombinant chimeric proteins able to redirect immune cell specificity and reactivity toward selected membrane antigens, and more particularly in which extracellular ligand binding is a scFV derived from a 5T4 monoclonal antibody, conferring specific immunity against 5T4 positive cells. The engineered immune cells endowed with such CARs are particularly suited for treating lymphomas and leukemia, and for solid tumors such as colon, stomach, and ovarian tumors.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a National Stage entry of International Application No.: PCT / EP2015 / 070128, filed Sep. 3, 2015, which claims priority to Danish Patent Application No. PA201470543, filed Sep. 4, 2014. The disclosure of the priority applications are hereby incorporated in their entirety by reference.FIELD OF THE INVENTION[0002]The present invention relates to Chimeric Antigen Receptors (CAR) that are recombinant chimeric proteins able to redirect immune cell specificity and reactivity toward 5T4, a cell surface glycoprotein found on most myeloid cells and used to diagnose solid tumors such as stomach, colon and ovarian tumors, and pre-B acute lymphocytic leukemia (ALL) in patients. The CARs according to the invention are particularly useful to treat malignant cells bearing 5T4 antigen, when expressed in T-cells or NK cells. The resulting engineered immune cells display high level of specificity toward malignant cells, conferring safety ...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K16/30C07K14/735C12N5/0783C12N5/078C07K19/00C07K14/725C07K14/705
CPCC07K14/7051C07K14/70578C07K14/70517C07K14/70535C07K14/705C07K19/00C07K16/30C12N5/0636C12N5/0634C07K2319/02C07K2319/74C07K2319/70C07K2319/33C12N2510/00C07K2317/622C07K2319/03A61P35/00A61P35/02A61P37/04A61K39/00118A61K2039/5156
Inventor SCHIFFER-MANNIOUI, CÈCILE
Owner CELLECTIS SA
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